The biotechnical revolution, including development of modern biopharmaceuticals and mapping of the human genome, has been made possible due to development of separation methods such as chromatography and electrophoresis. Such methods can be used in small scale as well as in large scale, and are known as flexible methods, being useful for a variety of substances including biological substances. However, they are demanding both technically and in terms of equipment. In addition, scaling of some processes such as electrophoresis results in a need for more complicated equipment due to nonlinear scaling of heating and cooling requirements.
Partitioning between the phases in aqueous polymer phase systems is an alternative method, which has been studied since the 1950's but whose commercial application has been severely restrained by lack of economically scalable phase systems. Together with separation methods such as crystallization and size exclusion; partitioning is considered a classic separation technique. It is related to differentially distributing a target and other substances between two phases. The term “partitioning” can refer to (a) liquid-solid partition such as in classic chromatography, (b) partitioning between two or more liquid phases (biphasic and multiphase system, respectively), (c) partitioning between a mobile liquid phase and another liquid phase immobilized at the surface of a solid phase support, and (d) partitioning of particles between a liquid phase and the phase interface between two phases. For the purposes of this patent application, partition and partitioning refer to situations such as b, c or d i.e. partitioning between liquid phases. Partition is typically expressed as a coefficient (K) related to the concentration in one phase versus another and for solutes K generally follows the Brønsted equation. Thus K is expected to vary exponentially with various types of interactions such as electrostatic and/or hydrophobic interactions, and also to be sensitive to solute size i.e. the area of interaction with liquid phases. In the case of interfacial partition, K is expected to vary exponentially with interfacial tension, which tends to localize particles to the phase interphase.
Classic two-phase systems are organic and aqueous two phase systems which normally have significant polarity differences between the phases, as well as significant interfacial tension. Such systems are not very useful for biologicals such as proteins or cells as they tend to be denatured by significantly apolar solutions and shear damage related to mixing of phase systems with significant interfacial tension. More useful for biologicals are the low tension, aqueous polymer two phase-systems. It is well recognized that the latter may contain some added organic solvents, e.g. ethanol, or other organic additives added to enhance target solubility, reduce liquid phase polarity, reduce foaming, act as bactericidal agents, etc.
Polymer two-phase systems can be formed by mixing certain hydrophilic and typically neutral polymers in aqueous solution. These include dextran (polyglucose) and poly(ethylene glycol) (PEG); as well as polysucrose (such as Ficoll™) and PEG; or linear polyacrylamide and PEG. Typical concentrations of each polymer are 5 to 10% w/w. At such concentrations, entropic forces tend to drive the formation of two phases both of which are typically greater than 90% (w/w) water but show subtle differences in polarity, hydrogen bond character, freezing point, etc. The phases are typically enriched in one polymer and have very low interfacial tension. In the biotechnical field, one advantage of the PEG and dextran type of two-phase system is that target proteins may partition in favour of the PEG-enriched phase while cell debris and some contaminants may partition to the interface or complementary phase.
WO 2004/020629 (Tjerneld) relates to the use of a PEG-like polymer comprising ethylene oxide (EO) groups in addition to propylene oxide (PO) groups, abbreviated as EOPO polymers. Such polymers, which show reverse thermal solubility, are known as “EOPO” polymers, and they are suggested in WO 2004/020629 for the separation of plasmids. At room temperature, the less dense, EOPO-enriched upper phase is isolated from the EOPO and dextran polymer aqueous two-phase system, and by a subsequent increase of its temperature to 37° C., the upper phase undergoes a further phase separation into a water-enriched phase and a self-associated EOPO polymer-enriched phase. Advantageously, the water-enriched phase should contain the desired plasmids. In general, this kind of EOPO and dextran systems offer advantages in terms of phase polymer component recycling and design of efficient two-stage partition separation process. However, a drawback is the cost involved in system formulation, which does not relate to the man-made synthetic polymer PEG but rather to the biologically derived and much more costly dextran.
Attempts to replace dextran with various starch or other polysaccharide polymers has resulted in limited success. One polymer, two-phase systems of intermediate interfacial tension can be formed by combining PEG and certain water structuring salts at relatively higher concentrations, e.g. 500 mM ammonium sulphate. PEG-salt two phase systems is one possible approach to overcome cost limitations but the increased PEG and salt concentrations create challenges which negatively impact process costs. These include viscous phases, salt reagent costs, salt disposal and equipment corrosion challenges, as well as target solubility issues which relate to capacity. As a consequence, the polymers are often difficult to recycle or otherwise have to be separated from the target via further downstream processing.
In the biotechnical field, polymer two phase systems, both in the forms with or without significant salt, are of general interest. This is because they are easily utilised in small as well as larger scale separations, without loss of efficiencies or dramatic changes in costs when scaling up to the larger volumes. Also, any standard separation approach, such as charge based, hydrophobicity based, affinity based, or size based separation, can be performed within a polymer two phase system. In general many undesired components, such as cell debris, endotoxins, nucleic acids will tend to appreciably partition to the lower (dextran-rich or salt rich, respectively) phase in a PEG and dextran or a PEG and salt two phase system. Thus, if a system can be found which provides for good target partition into the upper (PEG-rich) phase an effective primary separation and target concentration can be obtained.
Further, in efforts to overcome drawbacks related to interfacing in standard chromatographic and/or filtration processing, and to overcome the limitations of a single theoretical partition step per unit operation liquid-liquid partitioning two phase systems such as PEG-dextran or PEG-salt have been adapted to chromatographic uses by immobilising one phase on a chromatographic or other solid support capable of preferentially wetting that phase. The complementary phase is then pumped through the column offering repeated opportunities for equilibration between the mobile and stationary phase. This was commercially exploited by W. Müller et al. at Merck Darmstadt in the 1980's.
U.S. Pat. No. 5,093,254 (Giuliano et al) relates to an aqueous two-phase protein partitioning system is disclosed which employs polyvinylpyrrolidone as the upper phase and maltodextrin as the lower phase and provides a low-cost system for protein partitioning. The system can also be employed with the amion derivatives of chlorotriazine dyes, which bind in a noncovalent manner to the PVP and serve as a ligand for the proteins to be separated. It is stated that an advantage of this system is its cost-efficiency, as the dyes can easily be bound to the polymeric phase, without having to carry out the chromatographic and solvent extractions necessary to form the covalent bond in the PEG/hydroxypropyl starch system of the prior art. However, a drawback is the possible carcinogenic effect of such dyes.
Albertsson (P.-A. Albertsson, Partition of Cell Particles and Macromolecules, 2nd Edn., Wiley Interscience, N.Y., 1971. Chapter 10 Phase Diagrams, pp. 250-313) discloses systems comprising PEG and Na Carboxymethyl group modified dextran (CMD). The drawbacks of the system described is that (a) the polymer still involves an expensive polysaccharide; (b) the polymer is then further chemically modified; (c) the high molecular weight (Mw 2 200 000) and the inherent phase viscosity noted; and (c) the relatively high concentrations of polymers required to form phases, which is expected to bind water molecules and reduce system protein solubility.
Gupta et al (Vandana Gupta, Sunil Nath, Subhash Chand in Polymer 43 (2002) 3387-3390: Role of water structure on phase separation in polyelectrolyte-polyethyleneglycol based aqueous two-phase systems) relates to a study of the phase separation behaviour of polyelectrolyte-polyethyleneglycol (PEG) based aqueous two-phase systems (ATPS) carried out in order to elucidate the mechanism controlling phase-behaviour. Gupta uses polyethyleneimine (PEI) with a number average molecular weight of 60 000; and polyacrylic acid (PAA) with an average molecular weight of 250 000). It was concluded from this study that salt-assisted polymer-modified water structure interactions play a central role in phase separation in ATPS.
Saravanan (Settu Saravanan, Johny A. Reena, Jonnalagadda R. Rao, Thanapalan Murugesan, and Balanchandran U. Nair in J. Chem. Eng. Data 2006, 51, 1246-1249: Phase Equilibrum Compositions, Densities, and Viscosities of Aqueous Two-Phase Poly(ethylene glycol)+Poly(acrylic acid) Systems at Various Temperatures) relates to a study of the effect of temperature on the densities and viscosities of aqueous solution of poly(acrylic acid) (PAA) of different mass fraction (from 0.05-0.50) and liquid-liquid equilibrium, densities, and viscosities for the aqueous two-phase PEG-6000+PAA+water system at equilibrium.
As such there is still great need for novel separation methods, which are relatively technically simple and readily scaled.